Microneedles and uses thereof

Abstract

The present disclosure concerns a microneedle comprising a glycosaminoglycan (GAG) coating and / or a hydrogel layer on an external surface of the microneedle; wherein the microneedle and / or the hydrogel layer is porous.

Description

[0001] MICRONEEDLES AND USES THEREOF

[0002] Technical Field

[0003] The present invention relates, in general terms, to microneedles and their uses thereof.

[0004] Background

[0005] Inflammation-associated skin diseases such as diabetic ulcers and psoriasis are a significant healthcare challenge worldwide. Diabetic wounds affect 6.3% of people globally and are a leading cause of non-traumatic lower limb amputations with adverse impacts on quality of life. The defining characteristics of chronic diabetic wounds include persistent and uncoordinated inflammation which is mediated by an interplay between immune cell infiltration and chemokine secretion that contributes to impaired healing. Monocyte chemotactic protein-1 (MCP-1) is a chemokine that is present in high concentrations in wound tissues and is typically secreted in the first week post-wounding. It is a crucial signalling molecule orchestrating monocyte / macrophage recruitment. In particular, in response to heightened MCP-1 levels, blood monocytes, which originate from macrophage-dendritic cell precursors in the bone marrow, infiltrate into wound tissues and differentiate into pro-inflammatory Ml-like macrophages that further drive inflammation in wound sites. This creates a vicious cycle as newly infiltrated and differentiated inflammatory cells secrete more chemokines including MCP-1 to prolong wound inflammation. MCP-1 is also implicated in other inflammatory skin diseases such as psoriasis, where the MCP-1 produced by hyper-proliferated keratinocytes is a key mediator of monocyte infiltration, with monocyte-derived dendritic cells then aggravating skin inflammation.

[0006] Given the key role of chemokines in chronic inflammatory diseases, strategies to deplete chemokines from tissues have been deployed to inhibit infiltration of monocytes and alleviate inflammation. For example, systemic injection of anti- MCP-1 antibodies and topical application of chemokine-scavenging hydrogels improved wound healing by blocking the signalling functions of chemokines. While topical application of hydrogels has advantages over antibody therapy as it can deplete multiple chemokine species and circumvent the adverse side effects of systemic antibody administration, it is unable to sequester chemokines or remove pro-inflammatory monocytes spatially enriched in the wound bed, which is the root source of inflammatory chemokines. There is thus a need for improved methods of wound healing that can better neutralise the effects of proinflammatory chemokines at the wound site.

[0007] It would be desirable to overcome or alleviate at least one of the abovedescribed problems.

[0008] Summary

[0009] Disclosed herein is a microneedle comprising : a glycosaminoglycan (GAG) coating and / or a hydrogel layer on an external surface of the microneedle; wherein the microneedle and / or the hydrogel layer is porous.

[0010] In some embodiments, the GAG coating further comprises a polymer, wherein GAG is covalently bonded to the polymer.

[0011] In some embodiments, the GAG coating is a bilayer coating, comprising a first polymer layer adjacent to the microneedle, and a second layer comprising GAG covalently attached to the first polymer layer.

[0012] In one embodiment, the polymer is star polyethylene glycol (PEG).

[0013] In one embodiment, the GAG is a heparin. In some embodiments, the GAG is characterised by a concentration of about 1 mg / mL to about 5 mg / mL relative to the GAG coating.

[0014] In some embodiments, the hydrogel layer comprises gelatin crosslinked via a linker.

[0015] In some embodiments, the linker is a degradable linker.

[0016] In some embodiments, the microneedle comprises poly(lactic-co-glycolic acid) (PLGA).

[0017] In some embodiments, the microneedle is characterised by a pore size of about 5 pm to about 50 pm.

[0018] In some embodiments, the microneedle is characterised by a length of about 500 pm to about 1500 pm.

[0019] In some embodiments, the microneedle is characterised by a base width of about 400 pm to about 600 pm.

[0020] In some embodiments, the microneedle further comprises an active substance selected from an immunosuppressant, an antimicrobial compound, an antimetabolite, or a combination thereof.

[0021] In some embodiments, the microneedle is capable of recruiting a cytokine and / or a motile cell.

[0022] In some embodiments, the cytokine is a chemokine.

[0023] In one embodiment, the chemokine is monocyte chemoattractant protein-1 (MCP-1). In some embodiments, the motile cell is an immune cell.

[0024] In some embodiments, the immune cell is a monocyte or a macrophage.

[0025] Disclosed herein is a microneedle device, comprising a plurality of microneedles extending from a substrate, wherein one or more of the microneedles is a microneedle as defined herein.

[0026] In some embodiments, the plurality of microneedles is present as an array on the substrate.

[0027] In some embodiments, the distance between two microneedles on the microneedle array is about 500 pm to about 1000 pm.

[0028] In some embodiments, the substrate is attached to a dressing.

[0029] In some embodiments, the dressing comprises an adhesive for attaching to a tissue.

[0030] Disclosed herein is a method of treating a wound and / or a skin disease or condition in a subject in need thereof, the method comprising applying a microneedle device as defined herein to the wound and / or skin having the skin disease or condition such that one or more of the microneedles in the device penetrates the wound or skin.

[0031] In some embodiments, the microneedle recruits a cytokine and / or a motile cell from the wound.

[0032] In one embodiment, the wound is a chronic wound.

[0033] In one embodiment, the wound is a wound on a skin tissue. In some embodiments, the skin disease or condition is an inflammatory skin disease or condition.

[0034] In one embodiment, the skin disease or condition is psoriasis.

[0035] In some embodiments, the method further comprises contacting the microneedle device to the wound and / or skin disease or condition for at least about 12 h.

[0036] Disclosed herein is a method of isolating a chemokine and / or motile cell from a tissue from a subject, the method comprising (a) applying a microneedle device as defined herein to the tissue such that one or more of the porous microneedles in the device penetrates the tissue; and (b) removing the microneedle device from the tissue after a time sufficient for the porous microneedle to recruit the chemokine and / or motile cell from the tissue, thereby isolating the chemokine and / or motile cell from the tissue.

[0037] Brief description of the drawings

[0038] Embodiments of the present invention will now be described, by way of nonlimiting example, with reference to the drawings in which :

[0039] Figure 1 shows the preparation, characterization, and chemokine binding properties of heparin-coated porous microneedles (HPMN). A) Schematic illustrating sequestration of chemokines by HPMN, and attraction and removal of inflammatory monocytes / macrophages from the wound. B) Fabrication of HPMN . C) SEM images showing microscale pores distributed on the HPMN surface. Scale bar: 100 .m. D) Mechanical behavior of HPMN under compressive force and schematic of the experiment setup, n = 3. E) MCP-1 binding efficiency at various time points of HPMN. n = 3. F-H) Amount of chemokine bound to HPMN after 24 h at different initial chemokine amounts, including MCP-1 (F), MIP-lo (G), and IL-8 (H). n = 3. I) MCP-1 concentration change after BMDM- derived condition medium (CM) was incubated with HPMN. Yellow arrows indicate daily change of freshly prepared HPMN into CM, with HPMN incubated with CM for 24 h per condition, n = 3. *P < 0.05 and **P < 0.01.

[0040] Figure 2 shows that HPMN-depleted chemokines reduced monocyte chemotaxis and transmigration. A) Monocyte migration trace B) velocity, and C) directionality were evaluated by live cell tracking using pi-slide chambers, n = 3. D) Illustration of the transmigration assay. Relative migrated cells to the lower compartment of the Transwell for E) HPMN-treated MCP-1 and F) HPMN-treated CM showed that HPMN depleted MCP-1, thus reducing monocyte transmigration, n = 4. G) HPMN with enriched MCP-1 recruited monocytes. HPMN was labeled with rhodamine B isothiocyanate (RBITC, red), and monocytes were labeled with CellTrace CFSE (green). The number in the top left indicates the diameter of the cross-section of needles. White arrows indicate the presence of monocytes inside of HPMN. Scale bar: 100 |j.m. *P < 0.05, **P < 0.01, and ***P < 0.001.

[0041] Figure 3 shows that HPMN accelerates mouse diabetic wound healing. A) Schematic of experiment. Wounds were treated with HPMN or heparin-coated porous patch (HPP) on days 3-5. B) MCP-1 amount recovered from HPMN and HPP that were removed from wounds after 24 h of implantation, n = 10. *P < 0.05 of HPP compared with HPMN on day 3, and ***p < 0.001 of HPP compared with HPMN on day 5. C) Representative flow plots of Ly6c+cells (gated on CDllb+) and D) quantification analysis of Ly6C+CDllb+monocyte percentages in wounds showed HPMN treatment reduced wound infiltrated monocytes, n = 4. E) Representative images of wounds on days 0, 3, 7, 10, and 14, and schematic diagram of dynamic wound healing processes during 14 days for control groups, HPP-treated, and HPMN-treated groups, and F) quantitative analysis of relative wound area for each group on days 3, 7, 10, and 14 showed that HPMN accelerated wound healing, n = 10. G) Histological examination of wound sections including hematoxylin & eosin (H&E) staining, Masson's trichrome (MT) staining, immunofluorescence staining of CD31 (red), and immunohistochemistry staining of MCP-1 on day 14. Black dash lines indicate the wound edge. Statistical analysis of H) wound length from H&E-stained wounds, I) relative collagen intensity from Masson's trichrome-stained wounds, J) relative CD31 intensity from CD31 antibody-stained wounds, and K) relative MCP-1 intensity from MCP-1 antibody-stained wounds showed that HPMN treatment promoted wound closure, collagen deposition, and angiogenesis but reduced tissue MCP-1 level, n = 6. *P < 0.05, **P < 0.01, and ***p < 0.001.

[0042] Figure 4 shows that HPMN reduces tissue inflammation and promotes wound healing in a porcine wound model. A) Representative images of porcine wounds on days 0, 7, and 15 after treated with HPMN. Control referred to untreated wounds. B) H&E-stained wound sections and C) statistical analysis of wound width on day 15 showed HPMN treatment accelerated wound closure, n = 3. D) Immunofluorescence staining of CD31 (red) and semi-quantitative analysis of the blood vessel area showed HPMN treatment promoted angiogenesis, n = 3. E) Immunohistochemical detection of Ki67 and semi-quantitative analysis of the relative Ki67 intensity showed HPMN treatment improved tissue cell proliferation, n = 3. F) Immunohistochemical detection of Ibal (macrophages) and semi-quantitative analysis of the relative Ibal intensity, n = 3. G) Immunohistochemical detection of neutrophils and semi-quantitative analysis of the relative neutrophil intensity, n = 3. H) Immunohistochemical detection of CD3 (T cells) and semi-quantitative analysis of the relative CD3 intensity, n = 3. The combined F-H showed HPMN treatment reduced macrophages, neutrophils, and T cells presence in wound tissue. I) Relative expression of genes encoding pro-inflammatory cytokines and chemokines (dark grey columns), growth factors (dark blue columns), extracellular matrix structural components (dark red columns), and remodeling enzymes (orange columns) showed HPMN treatment down-regulated pro-inflammatory gene expression and up-regulated pro-healing gene expression. The values were normalized to control, n = 3. *P < 0.05 and **P < 0.01

[0043] Figure 5 shows that combination therapy of HPMN with MTX ameliorates psoriasis. A) Illustration of imiquimod-induced psoriasis model and MTX HPMN treatment. B) PASI scores were monitored for 7 days showed that MTX HPMN treatment attenuated psoriasis severity, n = 6. C) Picture of spleen and D) spleen weight on day 7 showed MTX HPMN reduced mice spleen weight than model group, n = 6. E) Mouse skin was imaged on day 7, and the skin was harvested for H&E staining showed decreased epidermis thickness in MTX HPMN treated mice. Scale bar: 100 urn. F) Immunohistochemical detection of Ki67 and G) semi-quantitative analysis of the relative Ki67 intensity showed MTX HPMN treatment inhibited keratinocyte over proliferation, n = 6. Scale bar: 100 p.m. H) Immunohistochemical detection of MCP-1 and I) semi-quantitative analysis of the relative MCP-1 intensity showed MTX HPMN treatment reduced the tissue MCP-1 level in psoriasis mice, n = 6. J-M) Statistical analysis of J) monocytes (Ly6c+in CDllb+), K) macrophages (CDllb+F4 / 80+in CD45+), L) dendritic cells (CDllc+in CD45+), and M) T cells (CD3+in CD45+) on day 7 after different treatment showed MTX HPMN treatment decreased monocytes, macrophages, dendritic cells, and T cells presence in psoriasis skin, n = 6. N) Relative mRNA expression of ILlb, IL17a, and IL23 on day 7 showed MTX HPMN treatment alleviated skin inflammation in psoriasis mice, n = 6. *P < 0.05, **P < 0.01, and ***P < 0.001.

[0044] Figure 6 is an SEM image of CaCOs microparticles. Scale bar: 10 p.m.

[0045] Figure 7 shows optimization of coating conditions of HPMN. A) Influence of StarPEG amount on MCP-1 binding efficiency. The molar ratio of heparin-to- StarPEG was kept at 1: 5. n = 3. B) Influence of heparin-to-StarPEG molar ratio on MCP-1 binding efficiency. The StarPEG amount was kept at 5 mg. n = 3.

[0046] Figure 8 shows indents caused by the penetration of microneedles into mouse skin. The skin was stained with trypan blue after HPMN application.

[0047] Figure 9 The MCP-1 amount that HPMN extracted from porcine wounds on day

[0048] 3 (D3) and day 7 (D7). n = 6. ***P < 0.001. Figure 10 shows A) MTX amount in HPMN before and after application to mouse skin showed ~50% MTX was delivered into mice skin, n = 4. B) Comparison of the MCP-1 binding efficiency of HPMN and MTX-loaded HPMN (MTX HPMN) showed MTX loaded on HPMN didn't affect HPMN sequestration of MCP-1. n = 3 ***p < o.OOl.

[0049] Figure 11 shows that MTX HPMN treatment reduced epidermal thickness of mice dorsal skin on day 7. n = 6. ***P < 0.001.

[0050] Figure 12 shows H-NMR characterization of synthesized Gelatin-Boc- Cystamine-MA (GSSMA).

[0051] Figure 13 shows degradation properties of GSSMA in the presence of different concentration of TCEP.

[0052] Figure 14 shows number of pore and size thereof of GSSMA hydrogel can be turned by using various dextran concentration.

[0053] Figure 15 shows GSSMA coated microneedles extracted and recovered murine macrophages (RAW 264.7) using an in vitro GelMA hydrogel model.

[0054] Detailed description

[0055] Modern microfabrication techniques enable the engineering of microneedle devices for use as advanced wound dressing and / or for minimally-invasive drug delivery, oxygen therapy, and cell therapy. Microneedles of different physical properties can be designed to penetrate various tissues to different depths for precision pharmacokinetics, detection of wound states (such as detecting pH and oxidative signals), and fluid extraction and sampling. The micron-scale dimensions of microneedle shafts allow for simple and direct application onto tissues like the skin without requiring professional training, and with minimal or no scarring, while causing significantly less pain than conventional hypodermic needles.

[0056] The inventors have engineered porous microneedles coated with glycosaminoglycans (GAGs) to alleviate inflammatory skin conditions and to treat wounds. Without wishing to be bound by theory, GAGs such as heparin can capture inflammatory cytokines through electrostatic interactions between negatively-charged sulphate groups on the GAGs and positively-charged amino acid residues on the cytokines. Chemokines are family of cytokines that guide cellular migration; many chemokines have a net positive charge. Advantageously, chemokine capture by GAGs on the microneedles can generate chemokine gradients around the microneedles to further recruit inflammatory cells.

[0057] In some embodiments, the inventors show that heparin-coated porous microneedles (HPMN) are able to penetrate tissue to spatially attract, concentrate and sequester multiple chemokines within the tissue, in particular the pro-inflammatory chemokine MCP-1. Without wishing to be bound by theory, heparin-mediated enrichment of MCP-1 on the microneedles can in turn recruit inflammatory cell types such as monocytes. The porous structure of the microneedles is advantageous in allowing infiltration and trapping of the recruited cells. Subsequent removal of the microneedles then depletes both inflammatory chemokines such as MCP-1 as well as their cellular source.

[0058] Additionally, the inventors have found that porosity may be introduced to the microneedle via a porous hydrogel coated on its surface thereof. As disclosed herein, a hydrogel coated microneedle was fabricated, in which the porous hydrogel coating material is formed from Gelatin-Boc-Cystamine-MA (GSSMA) and dextran. The coating material is UV crosslinkable, degradable and porous. The porous coating material allows penetration of cells.

[0059] The inventors show that the microneedles are broadly applicable for treating wounds as well as inflammatory skin conditions such as psoriasis. Additional therapeutic agents such as immunosuppressants and antimicrobial compounds can be incorporated in the microneedles to provide synergistic therapeutic effects. Furthermore, the microneedles can be used to extract cytokines and cells from tissues for quantitative analysis and monitoring of wound or disease progression.

[0060] Accordingly, the present disclosure concerns a microneedle comprising : a glycosaminoglycan (GAG) coating and / or a hydrogel layer on an external surface of the microneedle; wherein the microneedle and / or the hydrogel layer is porous.

[0061] Disclosed herein is a porous microneedle comprising a glycosaminoglycan (GAG) coating on an external surface of the microneedle.

[0062] As used herein, the term "porous" means having pores or voids throughout at least a portion of the microneedle or coating structure, sufficiently large and sufficiently interconnected to permit passage of fluid and solid materials. In the present context, the pores allow cells to pass through the microneedle and / or hydrogel layer.

[0063] The microneedles of this disclosure can be fabricated from a variety of materials, including metals and metal alloys (e.g., titanium, nickel, gold, molybdenum, chromium, cobalt, stainless steel, and the like), ceramics (e.g., silicon dioxide), semiconductors (e.g., silicon), polymers (natural or synthetic), and composites. The material should be sterilisable using standard methods.

[0064] In some embodiments, the microneedles are fabricated from a polymeric material. Suitable polymeric materials include but are not limited to polyesters such as polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL) and poly(lactic acid-co-caprolactone), polyanhydrides, poly(ortho)esters, polyurethanes, polyhydroxyalkanoates, polycarbonates, polyvinylpyrrolidone, polymethacrylic acid, ethylene-vinyl acetate, polytetrafluoroacetate, and polyoxymethylene. The polymer may be a biodegradable polymer, i.e., a polymer that is degradable in vivo.

[0065] In some embodiments, the microneedle comprises a polyester, such as polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL) and poly(lactic acid-co-caprolactone). In one embodiment, the polyester is poly(lactic-co-glycolic acid) (PLGA).

[0066] The porous microneedles can be fabricated using methods generally known in the art, such as by sintering, micro-moulding, phase separation, electrochemical etching, porogen leaching or additive manufacturing. Porogen leaching is advantageous for fabricating porous polymeric microneedles because of the simplicity in the modulation of porosity and pore size. Other methods of microfabrication are described in Bao et al. (Drug Deliv TransI Res. 2022; 12(2): 395-414), which is hereby incorporated by reference in its entirety. The skilled person may select a suitable fabrication method for a chosen material for the microneedle.

[0067] For example, the porous microneedles may be fabricated using CaCOs microparticles and PLGA. The CaCOs microparticles may be etched using an acid, such that a cavity or pore remains.

[0068] Generally, the porous microneedle should have the mechanical strength to penetrate a biological tissue and to remain intact during insertion, throughout placement on the tissue (e.g., for up to a few weeks), and during removal.

[0069] Alternatively, the microneedle may be a solid microneedle (without pores) and the pores may be introduced via a hydrogel layer. The hydrogel layer may be coated on an externally facing surface of the microneedle. The hydrogel may be a gelatin hydrogel. In this regard, gelatin is crosslinked. Thus, in some embodiments, either the microneedle or the hydrogel layer is porous.

[0070] The hydrogel may comprise gelatin, in particular Type A gelatin. Type A gelatin are obtained from acid-treated raw material, and has an isoelectric point at pH 6-9. In some embodiments, the gelatin has a gel strength of about 200 to about 500, or preferably about 300.

[0071] In some embodiments, the hydrogel is crosslinked via a linker. In some embodiments, the linker is cystamine methacrylate. The crosslinking density of the hydrogel may be characterised using, for example, NMR, gel migration patterns, and / or swelling characteristics. Alternatively, the crosslinking density may be experimentally obtained from rheology studies.

[0072] The hydrogel is porous. The pores of the hydrogel are created by mixing a sacrificial material with gelatin and curing the mixture, following which the sacrificial material is removed. The sacrificial material may be dextran.

[0073] In some embodiments, the amino moieties on gelatin is protected. For example, the amino moieties may be protected using tert-butyl carbamates. The linker may thus be covalently conjugated to the carboxylic moieties on gelatin.

[0074] In some embodiments, the linker and thus the hydrogel is degradable. For example, tris(2-carboxyethyl)phosphine may be used to reduce the disulfide bond in the cystamine methacrylate linker. The cells trapped within the hydrogel may thus be recovered for analysis.

[0075] In some embodiments, the hydrogel layer is characterised by a thickness of about 1 pm to about 10 mm, about 10 pm to about 10 mm, about 50 pm to about 10 mm, about 100 pm to about 10 mm, about 200 pm to about 10 mm, about 300 pm to about 10 mm, about 400 pm to about 10 mm, about 500 pm to about 10 mm, or about 1 mm to about 10 mm. The hydrogel layer is preferably of sufficient thickness in order for cells to penetrate and be retained.

[0076] The glycosaminoglycan (GAG) coating is formed as an external facing coating of the microneedle. The GAG coating is thus on top of the porous microneedle. The GAG coating may additionally be on top of the hydrogel layer. The GAG coating may cover a portion of or the whole of the external surface of the microneedle. For example, the coating may be at a tip portion of the microneedle, and / or a body portion of the microneedle. The coating may be substantially evenly distributed on the external surface of the microneedle. In preferred embodiments, the coating does not occlude or only minimally occludes the pores on the external surface of the microneedle, so that cells are still able to traverse the pores.

[0077] In some embodiments, the GAG coating and / or the hydrogel layer further comprises a polymer. GAG may thus be covalently bonded to the polymer to form the GAG coating. The hydrogel layer may be formed on top of the polymer layer.

[0078] A "polymer" herein may comprise monomer units that are the same or different. A "homopolymer" is a polymer having monomer units that are the same. A "copolymer" is a polymer having two or more different monomer units. Monomer units are "different" if they differ from each other by at least one atom or are different isomerically. Accordingly, polymers herein includes both homopolymers and copolymers. For example, the copolymer may be a random, alternating, block, graft copolymer. There is no particular limitation on the molecular weight of the polymer. The polymer may also be a straight chain polymer, a branched polymer, or a crosslinked polymer. For example, the polymer may be a star polymer, wherein the number of arms may be from 3 to 20.

[0079] The polymer may provide an interface for attaching the GAG and / or hydrogel to the microneedle. In such an instance, a suitable polymer is one that is capable of interacting with both the microneedle and the GAG. Thus, a suitable polymer may contain a functional moiety for covalently and / or physically attaching to the GAG and another same or different functional moiety for covalently and / or physically attaching to the microneedle. Alternatively or additionally, the polymer may provide an interface or have a functionality suitable for recruiting cytokines and / or cells to the microneedle. For example, the polymer may form a cross-linked network that is favourable for cell attachment.

[0080] The polymer may be a biocompatible polymer, i.e., a polymer that does not elicit a significant immune response or toxicity when in contact with living tissues. Biocompatible polymers include a variety of natural and synthetic polymers, examples of which include but are not limited to collagen, fibrin, hyaluronic acid, chitosan, alginate, cellulose esters and ethers (e.g., hydroxyethyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and the like), polyalkylene glycols (e.g., polyethylene glycol, polypropylene glycol and copolymers thereof), polyesters (e.g., polylactide, polyglycolide and co-polymers thereof), polyacrylates, polyacrylamides, polyurethanes, polyvinyl alcohol and silicones.

[0081] In one embodiment, the polymer is polyethylene glycol (PEG). In other embodiments, the polymer is a star PEG. PEG may be suitably functionalised so as to attach to the microneedle at a portion thereof and the GAG at another portion thereof. For example, the PEG may be functionalised with amino moieties. For example, the PEG may be 4-arm poly(ethylene glycol) amine (4- arm PEG-NHz).

[0082] In some embodiments, the polymer is crosslinked to form a polymer network. In some embodiments, the GAG coating comprises a polymer network. The polymer network may be a crosslinked polymer network. The polymer may be a component of a cross-linked polymer network. In this regard, the network may comprise at least another polymer. Cross-links in the polymer network may be covalent (e.g., chemical crosslinks), non-covalent (e.g., electrostatic interactions), or a mixture of both.

[0083] For example, the polymer may be 4-arm PEG-NH2. The amino functionalities between the 4-arm PEG-NHz may be crosslinked using dicarboxylic acid linkers to form a polymer network.

[0084] In some embodiments, the cross-linked polymer network is a hydrogel. A "hydrogel" refers to a polymer network capable of retaining a significant amount of aqueous fluid (such as at least 10% by weight of aqueous fluid). Hydrogels possess viscoelastic properties very similar to biological tissue due to their significant water content. Besides providing a conducive environment for cell or biomolecule recruitment, hydrogels may also provide a more natural interface between the microneedle and a biological tissue the microneedle is inserted in.

[0085] For example, the dicarboxylic acid linkers used to crosslink 4-arm PEG-NH? may be functionalised with hydrophilic moieties such that water molecules may be trapped or retained within the polymer network.

[0086] In some embodiments, the GAG coating and / or the hydrogel layer is a monolayered coating or a multilayered coating. When a monolayered coating is formed on the external surface of the microneedle, GAG or hydrogel may be present in the monolayer. When a multilayered coating is formed, each layer of coating may comprise a different polymer or polymer network. For example, a first polymer or polymer network in a first layer may be used for interfacing with the microneedle, and a second polymer or polymer network in a second layer may be used for interfacing with the patient when in use. The layers may be attached to each other covalently or via physical interaction. The GAG may be present only in the second layer. Having a multilayered coating may reduce adverse response from a patient, and may also allow for a controlled release of GAG and / or active ingredient (as disclosed herein). The GAG or hydrogel may be attached to the polymer covalently or non- covalently. In preferred embodiments, the GAG or hydrogel is covalently attached to the polymer, e.g., through hydroxyl, carboxyl and / or amino moieties on the GAG. Covalent attachment prevents the GAG or hydrogel from being released from the polymer, thus ensuring that any cytokine or cell that is recruited by the GAG remains on the microneedle.

[0087] In some embodiments, the polymer and / or polymer network is covalently attached to the microneedle surface. One or more external surfaces of the microneedle may be altered with a surface pre-treatment to facilitate attachment of the polymer. Typical surface pre-treatments include a variety of plasma treatments capable of altering surface functionality. For example, the microneedle may be treated with a nitrogen plasma to introduce amide functionalisation or with an oxygen plasma to introduce carboxylate functionalisation. The microneedle may also be chemically treated to form reactive functional groups on the surface of the material for attachment of the polymer.

[0088] For example, the GAG coating may be a bilayer coating, comprising a first PEG layer adjacent to or covalently attached to the microneedle, and a second layer comprising GAG covalently attached to the first PEG layer. The first PEG layer may be covalently attached to the microneedle, or physically attached to the microneedle.

[0089] In one embodiment, the polymer is an amine-functionalised polymer. GAGs can be conjugated to amine-functionalised polymers through carboxyl moieties on the GAG. Furthermore, amine-functionalised polymers can be reacted with suitably pre-treated polyester microneedles.

[0090] Glycosaminoglycans for use in the GAG coating may be isolated from natural sources or chemically synthesised. GAGs include natural isolates which have undergone chemical and / or enzymatic processing, such as depolymerisation, cleavage, sulphation or deacetylation. The GAG may be of any molecular weight. Suitable GAGs include but are not limited to heparin, heparan sulphate, chondroitin sulphate, dermatan sulphate, keratan sulphate, and hyaluronic acid. The GAG coating may contain a mixture of GAGs.

[0091] The thickness of the GAG coating can be measured using, for example ellipsometry, surface profilometry, interferometry, X-ray refl ecto m etry, atomic force microscopy, or ultrasonic thickness measurement. In some embodiments, the GAG coating is characterised by a thickness of about 1 pm to about 10 mm, about 10 pm to about 10 mm, about 50 pm to about 10 mm, about 100 pm to about 10 mm, about 200 pm to about 10 mm, about 300 pm to about 10 mm, about 400 pm to about 10 mm, about 500 pm to about 10 mm, or about 1 mm to about 10 mm. In some embodiments, the GAG coating is characterised by a thickness of about 1 pm to about 5 mm, about 1 pm to about 1 mm, about 1 pm to about 900 pm, about 1 pm to about 800 pm, about 1 pm to about 700 pm, about 1 pm to about 600 pm, about 1 pm to about 500 pm, about 1 pm to about 400 pm, about 1 pm to about 300 pm, about 1 pm to about 200 pm, or about 1 pm to about 100 pm.

[0092] In one embodiment, the GAG is a heparin. The heparin may be unfractionated heparin, low molecular weight heparin (such as heparin with an average molecular weight of between 4000 kDa and 6000 kDa, including but not limited to nadroparin, reviparin, enoxaparin, parnaparin, certoparin, dalteparin and tinzaparin), or ultra-low molecular weight heparin (such as heparin with an average molecular weight of less than 4000 kDa, including but not limited to semuloparin, bemiparin and fondaparinux).

[0093] In some embodiments, the heparin is present at a concentration of about 1 mg / mL to about 5 mg / mL relative to the coating. In other embodiments, the concentration is about 1.5 mg / mL to about 5 mg / mL, about 2 mg / mL to about 5 mg / mL, about 2.5 mg / mL to about 5 mg / mL, about 3 mg / mL to about 5 mg / mL, about 3.5 mg / mL to about 5 mg / mL, or about 4 mg / mL to about 5 mg / mL.

[0094] The presence of heparin on the microneedle surface can be determined using methods generally known in the art, such as contact angle measurement, energy dispersive x-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), Fourier Transform infrared spectroscopy (FTIR), Raman spectroscopy, ellipsometry, or time-of-flight secondary ion mass spectrometry (TOF-SIMS).

[0095] The microneedle can have a straight or tapered shaft. The shaft can have a circular cross-section in the perpendicular, or the cross-section can be noncircular. For example, the cross-section of the microneedle can be polygonal (e.g., square, triangular, etc.), oblong, trapezoidal, or another shape. Accordingly, the microneedle may be, for example, conical or pyramidal in shape.

[0096] As mentioned, the pores in the hydrogel may be formed using a sacrificial material. For example, dextran of molecular weight about 450,000 to about 650,000 may be used. Depending on the molecular weight and the relative amount, the size and number of pores may be controlled. For example, a mass ratio of gelatin to dextran may be about 8:2 to about 6:4, about 6: 1 to about 6:3, or about 6: 1 to about 6: 2.

[0097] The pores in the microneedles and / or the hydrogel are preferably sized to accommodate infiltration and sequestration of cells, particularly eukaryotic cells and more particularly motile mammalian cells. In some embodiments, the pore size of the microneedle and / or the hydrogel is about 5 pm to about 20 pm. For example, the pore size may be about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, or about 20 pm. Pore size can measured using electron microscopy. One of skill in the art can select the appropriate pore size required for specific applications. For example, one can adjust the pore size to permit passage of a particular solid material or cell type to be transported into or through the microneedle.

[0098] In some embodiments, the pore size of the microneedle and / or the hydrogel is about 5 pm to about 50 pm, about 10 pm to about 50 pm, about 15 pm to about 50 pm, or about 20 pm to about 50 pm.

[0099] In some embodiments, the porosity is characterised by a density of about 1 pore / mm3to about 30 pore / mm3. In other embodiments, the density is about 5 pore / mm3to about 30 pore / mm3, about 10 pore / mm3to about 30 pore / mm3, about 15 pore / mm3to about 30 pore / mm3, or about 20 pore / mm3to about 30 pore / mm3.

[0100] In some embodiments, the microneedle has a width at the base of about 400 pm to about 600 pm, such as a base width of about 400 pm, about 450 pm, about 500 pm, about 550 pm, or about 600 pm.

[0101] In some embodiments, the microneedle has a length of about 500 pm to about 1500 pm, such as a length of about 500 pm, about 600 pm, about 700 pm, about 800 pm, about 900 pm, about 1000 pm, about 1100 pm, about 1200 pm, about 1300 pm, about 1400 pm, or about 1500 pm.

[0102] The length of the microneedles may be suitably selected for a particular application, for example, to satisfy a penetration depth in a particular tissue.

[0103] In some embodiments, the microneedle, GAG coating and / or hydrogel layer further comprises an active substance. The active substance may be incorporated into the microneedle during or after fabrication. For example, microneedles may be soaked in a preparation of the active substance and dried so that the active substance forms a coat on the microneedle surface. The active substance may also enter the pores. The active substance may be a polypeptide, a polysaccharide, a polynucleotide, a lipid, an organic compound, an inorganic compound, or a combination thereof. Representative active substances include antimicrobials, anti-biofilm compounds, hormones, growth regulators, analgesics, immunosuppressants, anti-cancer compounds and anti-inflammatory compounds. In some embodiments, the active substance is an immunosuppressant or an antimicrobial compound.

[0104] For example, the active ingredient may be methotrexate. Methotrexate is in a class of medications called antimetabolites. Methotrexate treats cancer by slowing the growth of cancer cells. Methotrexate treats psoriasis by slowing the growth of skin cells to stop scales from forming. Methotrexate may treat rheumatoid arthritis by decreasing the activity of the immune system.

[0105] In one embodiment, the active substance is on an external surface of the microneedle. The active substance may be covalently or non-covalently attached to the GAG coating and / or hydrogel layer on the microneedle surface. In preferred embodiments, the active substance is releasable from the surface.

[0106] In some embodiments, the microneedle is capable of recruiting a cytokine and / or a motile cell. It was found that the negatively charged sulfate groups of heparin form electrostatic interactions with positively charged amino acid residues of cytokines to capture cytokines.

[0107] A skilled person can determine recruitment of cytokine or cell using known methods in the art. For example, cytokines may be identified by immunochemistry (using ELISA, Western blots, immunoprecipitation, etc.), protein function or activity assays, mass spectrometry, and the like. Cells may be identified using microscopy, immunofluorescence, genetic or molecular markers (e.g., cell surface markers), flow cytometry, functional assays, etc. In some embodiments, the cytokine is a chemokine. Chemokines are a family of small signalling proteins that play a crucial role in immune responses, inflammation, and cell migration. They are involved in the regulation of immune cell trafficking and activation. Examples of chemokines include but are not limited to Monocyte Chemoattractant Protein-1 (MCP-1 or CCL2); Macrophage Inflammatory Protein-la (MIP-la, or CCL3); Macrophage Inflammatory Protein- ip (MIP-ip, or CCL4); Regulated upon Activation, Normal T-cell Expressed and Secreted protein (RANTES, or CCL5); interleukin-8 (IL-8, or CXCL8); Stromal Cell-Derived Factor 1 (SDF-1, or CXCL12); Interferon-y Inducible Protein 10 (IP- 10, or CXCL10); Macrophage Inflammatory Protein-2 (MIP-2, or CXCL2); and Neurotactin (or CX3CL1).

[0108] In one embodiment, the chemokine is monocyte chemoattractant protein-1 (MCP-1).

[0109] The motile cell can be any cell type capable of independent movement, and in particular any cell type capable of chemotaxis (e.g., along a chemokine gradient). Examples of motile cells include but are not limited to immune cells (e.g., monocytes, macrophages, neutrophils, T cells, B cells, NK cells, NKT cells dendritic cells, eosinophils, and the like), fibroblasts, keratinocytes, stem cells (e.g., mesenchymal or tissue-specific stem cells), epithelial cells, and endothelial cells.

[0110] In one embodiment, the motile cell is an immune cell. In one embodiment the immune cell is a monocyte or a macrophage.

[0111] Disclosed herein is a method of fabricating a microneedle, comprising: conjugating a polymer and GAG on an external surface of the microneedle in order to form a GAG coating on the microneedle; and / or crosslinking gelatin in the presence of a sacrificial material on the external surface of the microneedle and subsequently removing the sacrificial material; wherein the microneedle and / or the hydrogel layer is porous. Disclosed herein is a microneedle device, comprising a plurality of microneedles extending from a substrate, wherein one or more of the microneedles is a microneedle as defined herein. In some embodiments, the microneedle device comprises an array of microneedles as disclosed herein.

[0112] The microneedles can be oriented perpendicular, substantially perpendicular, or at an angle to the substrate. Preferably, the microneedles are oriented perpendicular or substantially perpendicular to the substrate so that a larger density of microneedles per unit area of substrate can be provided.

[0113] In some embodiments, the plurality of microneedles is present as an array on the substrate. Microneedles in the array may be arranged in any desired pattern, such as uniformly spaced rows. The array of microneedles preferably includes microneedles of uniform orientation, dimensions and spacing, but can also include a mixture of microneedles having, for example, various orientations, lengths, diameters, cross-sectional shapes, and inter-needle spacing.

[0114] In some embodiments, the distance between two microneedles on the microneedle array is about 500 pm to about 1000 pm, such as about 500 pm, about 600 pm, about 700 pm, about 800 pm, about 900 pm, or about 1000 pm.

[0115] A microneedle device may comprise a patch in the form of a combination of an array, a pressure sensitive adhesive and backing. Microneedles may protrude from all or a portion of the microneedle array substrate surface.

[0116] The substrate of the device may be formed from a flexible material to allow the array or device to fit the contours of a tissue to which the array is to be applied, such as the skin. A flexible device will facilitate more consistent penetration during use, since penetration can be limited by deviations in the attachment surface. In some embodiments, the substrate is attached to a dressing. The dressing may be any clinically acceptable wound treatment article, including but not limited to woven and non-woven bandages, films, gels, foams, and composites of such.

[0117] In some embodiments, the dressing comprises an adhesive for attaching to a tissue. In preferred embodiments, the device is "user-friendly". For example, in transdermal applications, affixing the device to the skin should be relatively simple, without requiring special skills. A subject can press the device against the skin, ensuring that the needle penetrates the skin, and apply the adhesive to keep the device in place over the course of, for example, several days.

[0118] In some embodiments, the microneedle device comprises a plurality of microneedles extending from a substrate, wherein one or more of the microneedles is a porous microneedle as defined herein; wherein the substrate is an adhesive patch. The adhesive patch may be an adhesive film, such as Tegaderm™. The adhesive patch may be sized to be larger than the microneedle array. The plurality of microneedles may be a microneedle array. The microneedle array may comprise a first surface having the microneedles, and a second surface opposite the first surface. The second surface of the microneedle array may be adhered to the adhesive patch. As the adhesive patch is sized larger, an adhesive border is formed. This may aid in attaching the microneedle device to a patient, following which the microneedle device may be activated by pressing the microneedle device into a skin of the patient such that the microneedle array penetrates a surface of the skin.

[0119] Disclosed herein is a method of treating a wound in a subject, the method comprising applying a microneedle device as defined herein to the wound such that one or more of the porous microneedles in the device penetrates the wound tissue. As used herein, the term "wound" refers to an injury to a tissue. Wounds include both open wounds (in which the underlying tissue is exposed to the outside environment, such as, for example, a laceration, puncture, burn or surgical incision) and closed wounds (in which the underlying tissue is not exposed to the outside environment, such as, for example, pressure sores, wounds induced by blunt trauma, and wounds caused by surgical implants). The wound may be an acute or a chronic wound.

[0120] In one embodiment, the wound is an acute wound. An "acute wound" herein is an injury which occurs rapidly (such as a cut, laceration, contusion, burn, etc.) which typically heals quickly and is expected to move through the normal stages of the healing process at the expected rate, ultimately resulting in complete closure of the wound. Wounds can display a spectrum of healing rates, whereby acute and non-healing wounds lie at opposite ends of the spectrum. Acute wounds may be expected to heal within three months. The skilled person will be able to determine expected timeframes for wound healing based on, for example, the severity of the wound, the site of the wound, the type of wound (e.g., open or closed), and the age and health condition of the wounded subject. Acute wounds can happen anywhere on the body and range from superficial scratches to deep injuries that damage the blood vessels, nerves, and muscle tissue.

[0121] In one embodiment, the wound is a chronic wound. A "chronic wound" herein is a non-healing or slow-healing wound that fails to progress through the usual phases of healing in an orderly way or at the expected rate. Wounds that do not heal within three months, for example, are considered chronic. A chronic wound may be characterised at least in part by one or more of (1) a prolonged self- perpetuating state of wound inflammation; (2) a deficient, defective and / or slow-forming wound extracellular matrix; (3) poorly responding (senescent) cells at the wound site, especially fibroblasts, limiting extracellular matrix production; and (4) failure or a decreased rate of re-epithelialisation or wound closure. Chronic wounds may be slow-healing wounds that heal at a slower rate than expected but still demonstrate some healing over time. Chronic wounds may also be non-healing wounds which do not show improvement or closure despite appropriate care.

[0122] Non-limiting examples of chronic wounds include ulcers, such as pressure ulcers (also known as decubitus ulcers), diabetic ulcers, diabetic foot ulcers, arterial ulcers, venous ulcers, venous stasis ulcers, vasculitic ulcers, burn ulcers, trauma -induced ulcers, infectious ulcers, and pyoderma gangrenosum ulcers. Chronic wounds include recurrent wounds caused by chronic skin or epithelial conditions such as acne, psoriasis, atopic dermatitis and keratitis. Chronic wounds also include non-healing or slow-healing surgical wounds, including dehiscent wounds, which are wounds, usually stitched or stapled surgical incisions, that have ruptured or split open.

[0123] In one embodiment, the wound tissue is skin tissue. The wound may affect any layer of the skin tissue, including the epidermal, dermal and / or subcutaneous skin layers. The porous microneedle should preferably penetrate beyond the epidermal layer of the skin to access the dermal layer.

[0124] In some embodiments, the subject is suffering from a skin disease or condition.

[0125] In one embodiment, the skin disease or condition is an inflammatory skin disease or condition. The inflammatory skin disease or condition may be, for example, psoriasis, eczema, dermatitis (e.g., contact dermatitis, seborrheic dermatitis, atopic dermatitis), xerosis, epidermolytic hyperkeratosis, ichthyosis, acne, folliculitis, keratoses, pruritis, dermatoses, urticaria or vitiligo.

[0126] In one embodiment, the skin disease or condition is psoriasis.

[0127] In some embodiments, the porous microneedle recruits a cytokine and / or a motile cell from the wound. The microneedle array may be kept in place on the tissue for a predetermined period of time (e.g., for at least 12 h, or 12 to 24 hours, or for several days) to allow recruitment and depletion of the cytokine and / or cell from the tissue.

[0128] In one embodiment, the method further comprises removing the microneedle array from the wound tissue. A new microneedle array may be re-applied to the wound or tissue to continue treatment. Removal and re-application of a microneedle array may be performed multiple times depending on the severity of the wound and the desired level of treatment.

[0129] Disclosed herein is a method of treating a skin disease or condition in a subject, the method comprising applying a microneedle device as defined herein to the skin of the subject such that one or more of the porous microneedles in the device penetrates the skin tissue.

[0130] Disclosed herein is a method of isolating a chemokine and / or motile cell from a tissue from a subject, the method comprising (a) applying a microneedle device as defined herein to the tissue such that one or more of the porous microneedles in the device penetrates the tissue; and (b) removing the microneedle device from the tissue after a time sufficient for the porous microneedle to recruit the chemokine and / or motile cell from the tissue, thereby isolating the recruited chemokine and / or motile cell from the tissue.

[0131] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0132] As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

[0133] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0134] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0135] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0136] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

[0137] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0138] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

[0139] Examples

[0140] Materials and methods

[0141] Carboxy-terminated poly (lactic-co-glycolic acid) (PLGA, molecular weight: 80 kDa) was purchased from Jinan Daigang Biomaterial Co., Ltd. 4-arm Poly(ethylene glycol) amine (4-arm PEG-NH?) was purchased from Xiamen Sinopeg Biotech Co., Ltd. Heparin sodium salt from porcine intestinal mucosa, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N- hydroxysuccinimide (NHS), ), lipopolysaccharides (LPS), fetal bovine serum (FBS), Liberase TM, collagenase D, gelatin type A from porcine skin, dextran (Mr 450,000-650,000), and hyaluronidase were purchased from Merck. Methotrexate was purchased from Tokyo Chemical Industry (TCIStreptozotocin and trypan blue were purchased from Aladdin Scientific. Recombinant murine MCP-1, recombinant murine MIP-a, recombinant human IL-8, recombinant murine M-CSF, and murine MIP-a ABTS ELISA development kits were purchased from PeproTech. Mouse MCP-1 ELISA kits, human IL-8 ELISA kits, Zombie Violet Fixable Viability Kit, and all the antibodies used for flow cytometry study were purchased from BioLegend. RPMI 1640 medium, Opti-MEM, penicillinstreptomycin, sodium pyruvate, 2-mercaptoethanol, MEM non-essential amino acids, CellTrace CFSE Cell Proliferation Kit, and Porcine CCL2 / MCP-1 ELISA Kit were purchased from Thermo Fisher. Mouse peripheral blood monocyte isolation kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0142] Fabrication of porous microneedles CaCOs microparticles were prepared by mixing equal volumes of NazCOs (0.33 M) and CaCh (0.33 M) and stirring at room temperature for 1 h, then the microparticles were washed with distilled water and lyophilized for further use. PLGA (2 g) was dissolved in 10 mL dioxane, then CaCCh microparticles (2 g) were thoroughly mixed with PLGA solution. The CaCCh microparticle-containing PLGA solution was added to microneedle molds and placed under a vacuum. The mold was then dried at room temperature overnight and heated at 70°C for 24 h, and the PLGA microneedles were peeled off from the mold. To etch CaCCh microparticles, PLGA microneedles were added to hexane / HCI solution (v:v = 1 : 1) and incubated for 2 h. The obtained porous microneedles were then washed thoroughly with distilled water and dried at 37°C. The porous microneedle patch consisted of needles with a height of 1000 gm, base diameter of 420 p.m, and center-to-center interval of 750 |_im.

[0143] Fabrication of heparin-coated porous microneedles (HPMN)

[0144] The porous microneedles were first hydrolyzed under 0.05 M NaOH solution for 10 min. After washing with distilled water, the PMN was immersed into a 0.5 mL MES buffer (50 mM, pH 6) containing 40 mM EDC and 20 mM NHS at 4 °C and incubated for 12 h. The EDC / NHS-activated porous microneedles were then incubated with StarPEG or 4-arm PEG-NH2 at 4°C for 5 h. Meanwhile, heparin was dissolved in MES buffer and activated by reaction with EDC and NHS at 4°C for 5 h. Next, EDC / NHS-activated heparin was added to the wells at incubated for 5 h. Finally, the HPMN was washed thoroughly with distilled water for 30 min thrice, dried at 37°C for 12 h, and stored at 4°C. Morphology of HPMN was viewed using a scanning electron microscope (JSM-6701F FEG, JEOL). The mechanical property was tested using a compression testing machine (EZ-SX, SHIMADZU) at a speed of 1.2 mm / min, the force began to be recorded upon the sensor contact with the top of the needles.

[0145] Fabrication of methotrexate-loaded HPMN

[0146] Methotrexate (MTX) was dissolved in MES buffer (50 mM, pH 7.4) at the concentrations of 0.5 mg / mL. MTX-loaded HPMN (MTX HPMN) was fabricated by incubating HPMN with 0.5 mL MTX solution for 1 h, then the microneedles were taken out and dried at 37°C. To test the MTX loading content in HPMN, the HPMN was immersed into 0.5 mL MES buffer and incubated for 2 h, after that, MTX concentrations were detected by UV-vis spectrometer (Evolution 201, Thermo Fisher) at a wavelength of 302 nm. To evaluate the transdermal drug delivery efficiency, MTX HPMN was inserted into the skin of mice and mounted on the skin using 3M Tegaderm transparent dressing. After 12 h, the MTX HPMN was taken off and immersed in fresh MES buffer to dissolve the remaining MTX.

[0147] In vivo skin piercing

[0148] Male Balb / c mice (6-8 weeks) were purchased from the laboratory animal center of Sun Yat-sen University. All the mice experiments were conducted by the protocol approved by the Ethics Committee of the School of Life Science, Sun Yat-sen University. After pressing HPMN on mice's skin for 10 min, the HPMN was removed, and the skin was stained with trypan blue. After 20 min of waiting, the skin was imaged using a digital camera.

[0149] Chemokine binding

[0150] HPMN was incubated with 1 mL R.PMI medium containing 2% FBS and 1000 ng / mL MCP-1 in a cell incubator. At 0, 1, 2, 4, 8, 12, and 24 h post incubation, mediums were taken out from the medium and stored under -80°C until detected by ELISA. To test the binding capacity of chemokines, HPMN was incubated with mediums containing 10, 100, or 1000 ng chemokines for 24 h and then the chemokine concentration was evaluated by ELISA assay.

[0151] Preparation of bone marrow-derived macroohaoes (BMDMs)

[0152] BALB / c mice were euthanized, and bone marrow was isolated. The bone marrow was suspended in a complete RPMI medium that consisted of RPMI 1640 medium, 10% heat-inactivated FBS, 1% penicillin-streptomycin, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, 1% MEM non-essential amino acids, and 20 ng / mL of freshly thawed M-CSF, and then seeded in non-tissue culture treated 24-well plates at 0.5 x 106cells / welL The complete RPMI medium was replaced every 3 days, and bone marrow-derived macrophages (BMDM) were obtained after the monocytes were cultured in the complete RPMI medium for 7 days.

[0153] Macrophage conditioned medium

[0154] BMDMs were cultured with RPMI 1640 medium containing 2% FBS, 1% penicillin-streptomycin, and 100 ng / mL of LPS. After 24 h, the cell-free BMDM- derived condition medium (CM) was collected and stored at -80°C. To test the MCP-1 binding capacity in BMDM-derived CM, fresh HPMN was added into CM on days 0, 1, and 2, after 24 h of incubation, the HPMN was taken out and the remaining MCP-1 in the medium was determined by ELISA assay. The CM without HPMN treatment was as control. The CM was collected on days 1, 2, and 3 for further use, and named CM + HPMN DI, CM + HPMN D2, and CM + HPMN D3, respectively.

[0155] Directional migration assay

[0156] Peripheral blood monocytes were isolated using mouse peripheral blood monocyte isolation kits per instructions provided by the manufacturer. Monocytes were seeded into the center of pi-slide chambers (ibidi) at a density of 3 x 106cells / mL and incubated for 30 min. After that, RPMI 1640 medium was filled into the left chamber, and RPMI 1640 medium, MCP-1 medium (10 ng / mL), or MCP-1 medium that had been pre-treated with HPMN for 24 h was filled into the right chamber. The cell migration track was captured every 20 min over 6 hours. Cell tracking analysis was carried out by the Manual Tracking plugin for Image! and the Chemotaxis and Migration Tool according to the protocol provided by ibidi.

[0157] Monocyte transmigration was carried out using Transwell chambers (3 |_im pore size, Corning). Briefly, monocytes (2 x 105cells) suspended in 0.1 mL of RPMI 1640 medium were added to the upper compartment of the Transwell chambers, and the lower wells were filled with RPMI 1640 medium containing MCP-1 (10 ng / mL) with or without HPMN treatment. Or the lower wells were filled with CM or the HPMN-treated CM (CM + HPMN DI, CM + HPMN D2, and CM + HPMN D3). After 2 h, the monocytes that migrated into the lower wells were counted under microscopy.

[0158] Monocyte migration into HPMN

[0159] To label HPMN with fluorescence dyes, Rhodamine B isothiocyanate (RBITC) was mixed in PLGA solution before casting into microneedle molds. RBITC labeled HPMN was first incubated with MCP-1 (10 ng / mL) contained medium for 24 h, then transferred to MCP-l-free culture medium and placed in the lower well of the Transwell. Freshly isolated monocytes (2 x 105cells) were stained with CellTrace CFSE and added to the upper compartment of the Transwell. After 12 h, the HPMN was taken out and transferred to a paraformaldehyde solution- contained glass well, and the cells that migrated into HPMN were visualized under a confocal laser scanning microscope (CLSM, Leica).

[0160] Mouse diabetic wound model

[0161] The diabetic mice were established by intraperitoneal injection of streptozotocin (STZ, 50 mg / kg body weight) solution for 5 consecutive days. Mice with fasting blood glucose levels higher than 16.7 mM for 2 successive days were regarded as diabetic mice. To induce the diabetic wound model, the diabetic mice were anesthetized with isoflurane, and the back area was shaved, two full-thickness cutaneous wounds in each mouse were made using an 8 mm round skin biopsy punch (day 0), and all the wounded mice were covered with 3M Tegaderm transparent dressing. The diabetic wound mice were randomly divided into three groups: diabetic mice without any treatment were as control; diabetic mice were treated with heparin-coated patches without needles (HPP) at days 3, 4, and 5 post wounding; and diabetic mice were treated with HPMN at days 3, 4, and 5 post wounding. The HPP and HPMN were taken away from the wound after 24 h of implantation and put into a 48-well plate containing 0.2 mL of 1% Triton X- 100 and incubated on ice for 5 min to recover the captured MCP-1, the solution containing MCP-1 was then stored under -80°C until further analysis by ELISA. The wounds were imaged on days 0, 3, 7, 10, and 14. Wound samples were excised from mice on day 14 and fixed using 4% paraformaldehyde.

[0162] Porcine wound model

[0163] All the pig experiment was approved by the Institutional Animal Care and Use Committee, National University of Singapore. The female Landrace cross pig weighing 50 kg was purchased from the Singapore National Large-Animal Research Facility. The pig was anesthetized by intramuscularly premedicated with lO mg / kg of ketamine, 0.04 mg / kg of atropine, and 0.6 mg / kg of midazolam, then induced with 4% isoflurane, and intubated maintained with 1- 2% isoflurane. Porcine dorsal skin was shaved and disinfected with 70% ethanol and betadine before producing full thickness 2 x 1 cm excisional wounds. All wounds were covered with 3M Tegaderm transparent dressing, and an elastic bandage was used to encircle the body. The wounds in HPMN treatment group received two 1 x 1 cm HPMN in each wound on days 3 and 7 post wounding, wounds without ant treatment were control. The experiment was terminated on day 15, and wound samples were collected for histological analysis and realtime quantitative PCR assay.

[0164] Psoriasis model

[0165] The imiquimod-induced psoriasis mouse model was established by topical treatment of imiquimod cream. Briefly, the back area of the mice was shaved and depilated one day before the induction, then the delimited back skin was topically administrated with 5% imiquimod cream (62.5 mg per mouse, Mingxin Pharmaceuticals) once a day for 6 consecutive days. The mice were randomly divided into 4 groups: normal mice without any treatment; psoriasis mice without any treatment; and psoriasis mice treated with HPMN. The microneedles (four of 1 x 1 HPMN) were applied to the back area and fixed on the skin for 12 h using 3M Tegaderm transparent dressing from day 3 to day 6. Mouse body weight and psoriasis area and severity index (PASI) were recorded during the treatment. The PASI was evaluated according to the sum score of erythema, scaling, and induration. Each symptom was scored as follows: 0, no symptom; 1, mild; 2, moderate; 3, severe; and 4, very severe. The mice were sacrificed at the end of the experiment, spleens were taken out and weighted, and the skin was collected for histological analysis and real-time quantitative PCR assay.

[0166] Flow cytometry analysis

[0167] Skin tissues from mice were harvested using a skin biopsy punch, and the attached fat tissue was removed. The skin sample was diced and digested using Opti-MEM containing 100 fig / mL Liberase TM, 1 mg / mL collagenase D, and 1 mg / mL hyaluronidase at 37°C for 30 min. The digested samples were then flushed through 70-p.m cell strainers using a syringe plunger. The blood cells in the cell suspension were then lysed with red blood cell lysis buffer and washed twice with PBS. The cells were stained with Zombie Violet Fixable Viability Kit, followed by blocking nonspecific binding by the incubation of anti-CD16 / 32 for 30 min. After that, the cells were stained with anti-CD45, anti-CDllb, anti- Ly6C, anti-F4 / 80, anti-CDllc, and anti-CD3 antibodies for another 30 min, then washed and analyzed by flow cytometry.

[0168] Real-time quantitative PCR

[0169] Total RNA was extracted from the skin using E.Z.N.A Total RNA Kit (Omega) according to the manufacturer's instructions. RNA quantity and quality were tested using Nanodrop (Thermo Fisher). Complementary DNA (cDNA) was synthesized by the iScript cDNA Synthesis Kit (BIO-RAD). Real-time quantitative PCR was performed with a StepOnePlus™ Real-Time PCR System (Applied Biosystems) using PowerTrack SYBR Green Master Mix (Thermo Fisher). The primers used for porcine study were purchased from Integrated DNA Technologies, Inc. and listed in Table 1, and primers used for mice study were purchased from Sangon Biotech and listed in Table 2.

[0170] Histological examination

[0171] The fixed skin tissues were embedded in paraffin and then cut into 5 gm sections, the deparaffinized and rehydrated sections were stained with hematoxylin & eosin (H&E) and Masson's trichrome (MT) staining and observed using a Vectra Automated Quantitative Pathology Imaging System (PerkinElmer).

[0172] Immunofluorescence and immunohistochemistry staining

[0173] The deparaffinized and rehydrated sections were immersed in 10 mM sodium citrate buffer and heated using a microwave for antigen recovery. Then the sections were blocked with 10% goat serum for 2 h at room temperature. After that, the sections were covered with primary antibodies and incubated at 4°C overnight. The primary antibodies include rat anti-CD31 (#ab7388, Abeam), rabbit anti-MCP-1 (#ab7202, Abeam), rabbit anti-Ki67 (#abl5580, Abeam), rabbit anti-Ki67 (#27309-l-AP, Proteintech), rabbit anti-Ibal (#019-19741, FUJIFILM), mouse anti-neutrophils (#T-3503, BMA Biomedicals), and rabbit anti-CD3 (#17617-1-AP, Proteintech). For immunofluorescence staining, the sections were stained with Alexa Fluor 488-conjugated (Abeam) secondary antibodies at room temperature for 1 h, followed by counterstaining with DAPI and then observed under a fluorescence microscope. For immunohistochemistry staining, the sections were pre-incubated with 0.3% H2O2 for 15 min before blocking with 10% goat serum, after primary antibodies staining, the sections were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Abeam) for 1 h. Finally, the sections were treated with DAB substrate (Abeam) and counterstained with hematoxylin for the following observation.

[0174] Statistical analysis

[0175] Quantitative results were presented as mean values ± SD, and statistical analyses were conducted using unpaired two-tailed Student's t-test by GraphPad software (Version 8.0, GraphPad Software). *P < 0.05, **P < 0.01, ***p < 0.001,#P < 0.05, and###P < 0.001 were considered statistically significant.

[0176] Example 1: Fabrication of heparin-coated porous microneedles (HPMN) HPMN was fabricated by micro-molding using a mixture of PLGA and CaCOs microparticles (~5 j m) (FIG. 6) as the casting solution. PLGA was selected as the microneedle matrix material to provide stable mechanical strength, and CaCOs microparticles were used to create porous surfaces with the etch of hydrogen chloride (Step 1). After etching, the PLGA chains on the surfaces of porous microneedles were hydrolyzed by sodium hydroxide to expose the carboxyl groups for subsequent EDC / NHS activation (Step 2). Finally, carboxyl groups on microneedles were reacted with amines on StarPEG to immobilize the heparin / StarPEG network on the surfaces of microneedles (Step 3) (FIG. IB). The coating condition was optimized for MCP-1 binding, with an efficiency of 83.7 ± 2.0% at a heparin-to-StarPEG molar ratio of 1 : 5 (FIG. 7). Scanning electron microscope (SEM) showed that micropores larger than 5 p.m were distributed on the surfaces of conical-shaped microneedles due to etching of CaCCh microparticles (FIG. 1C). Each needle could withstand a compression force of 0.7 N at the displacement of 0.5 mm which was previously reported to be sufficient to insert into skin without needle breaking (FIG. ID) and further confirmed in vivo using mouse skin with trypan blue indicators (FIG. 8).

[0177] Example 2: HPMN sequesters chemokines and reduces monocyte chemotaxis and transmigration

[0178] Heparin was chosen as the coating material for microneedles as the negatively charged sulfate groups of heparin are known to form electrostatic interactions with positively charged amino acid residues of chemokines to capture chemokines. From the binding kinetics of MCP-1 as shown in FIG. IE, it was observed that MCP-1 quickly bound to HPMN during the first 12 h and reached a plateau thereafter. The binding capabilities of HPMN were also tested for different chemokine species including MCP-1, macrophage inflammatory protein 1 alpha (MIP-la), and interleukin-8 (IL-8) at various amounts (FIG. 1F-H). MlP-la, a monocyte / neutrophil chemoattractant, and IL-8, a neutrophil chemoattractant were chosen as they are associated with prolonged immune cell infiltration and inflammation. There was a strong correlation between the initial chemokine amounts (10-1000 ng) and its relative binding amounts. MCP- 1 was found to bind the best to HPMN (81.6 ± 0.3%), followed by MIP-lo (27.6 ± 4.1%), and IL-8 (24.5 ± 6.4%) at the same initial chemokine amount of 1000 ng.

[0179] A proposed use of HPMN is to sequester and reduce chemokine gradients from chemokine-enriched wound sites via the removal of HPMN . The differentiation of macrophages (BMDM) from bone marrow-derived monocytes was first induced, followed by activation with LPS to induce proinflammatory chemokines production. The conditioned media (CM) from inflammatory macrophages is known to contain a complex mixture of pro-inflammatory secretomes. After incubation of CM with HPMN on days 0, 1, and 2 (with a daily change in HPMN), a significant decrease in MCP-1 levels was observed in CM (FIG. II), thus supporting the earlier observations that while HPMN could sequester different chemokines, it preferentially binds to MCP-1.

[0180] MCP-1 plays a major role in regulating the migration and infiltration of monocytes from the bloodstream into tissues, thus depletion of MCP-1 could reduce monocyte recruitment. Using a jx-sl ide chemotaxis assay, it was observed that, compared to only having MCP-1, the addition of HPMN (MCP-1 + HPMN) significantly reduced the migration velocity and directionality of primary murine monocytes (FIG. 2A-C). Next, using a transmigration assay with monocytes placed in the upper compartment, it was observed that the MCP-1 medium pretreated with HPMN (MCP-1 + HPMN) reduced the number of migrated monocytes into the lower well by 52 ± 7.3% relative to having MCP-1 solution alone (FIG. 2D-E). HPMN-treated CM was collected on day 1, 2 and 3, and named CM + HPMN DI, CM + HPMN D2, and CM + HPMN D3, respectively, and were evaluated for their ability to induce monocyte migration using a transmigration assay. HPMN-treated CM significantly decreased monocyte migration and the relative migrated cell percentages were 68.7 ± 3.9%, 55.3 ± 11.4%, and 45.0 ± 8.2% for CM + HPMN DI, CM + HPMN D2, and CM + HPMN D3, respectively, which was in consensus with the observed daily decrease in MCP-1 amounts shown in FIG. 1H (FIG. 2F).

[0181] The depletion of MCP-1 in solution may be attributed to the sequestration and enrichment of MCP-1 into HPMN which would have generated chemokine gradients to recruit monocytes into HPMN. To visualize the migration of monocytes into HPMN, monocytes and HPMN were labeled with CellTrace CFSE and rhodamine B isothiocyanate (RBITC), respectively, and monocyte distribution was captured using the confocal microscope. As observed in FIG. 2G, green dots indicative of monocytes were scattered throughout the red circle indicating infiltration of monocytes (white arrows) into HPMN. This data strongly suggests that monocytes could be spatially depleted from inflammatory tissues by removing HPMN with infiltrated monocytes.

[0182] Example 3: Application of HPMN accelerates wound healing

[0183] To assess the therapeutic effects of HPMN on inflammatory skin conditions, a mouse model of diabetic wound was used, and HPMN was compared against a heparin-coated porous patch (HPP) without the needle array as a benchmark to evaluate the therapeutic utility of spatial sequestration of chemokines in deeper diabetic wound tissues against topical application of a chemokine-sequestering material. HPMN and HPP were daily applied to diabetic wounds on days 3-5 (FIG. 3A). HPMN and HPP were taken out from wounds 24 h after application, and the captured MCP-1 was recovered for ELISA quantification. Both HPP and HPMN sequestered large amounts of MCP-1 from inflammatory diabetic wounds, thus surface functionalization of heparin was effective in scavenging chemokines under pathological conditions of diabetic wound inflammation (FIG. 3B). HPP extracted a similar quantity of MCP-1 on days 3, 4, and 5. Notably, the quantity of MCP-1 recovered from HPMN steadily decreased from day 3 to day 5. The MCP-1 concentration was 49.8 ± 26.7% and 21.1 ± 12.2% on day 4 and day 5, relative to day 3, respectively. Compared to HPP, on day 3, HPMN extracted greater amounts of MCP-1 which could stem the positive feedback loop between chemokines and inflammatory immune cells. It was hypothesized that the lower quantities of extracted MCP-1 amount by HPMN on day 4 and 5 could be attributed to a reduction of local tissue concentrations of MCP-1 after extraction of inflammatory immune cells from deep tissue layers on day 3. To test this hypothesis, wound tissues were digested and the number of activated Ly6c+monocytes were quantified. It was found that HPMN treatment remarkably decreased the monocyte levels by 23.9 ± 3.7% on day 7 and 42.8 ± 12.3% on day 14 compared to untreated wound control (FIG. 3D).

[0184] Compared to untreated control, HPMN-treated wounds had a faster wound closure rate and 47.1 ± 19.2% smaller open wound area by day 14 (FIG. 3E, F). Wound healing using HPMN was also better than HPP by 21.9 ± 28.4%. H&E- stained wound sections found that HPMN treatment improved wound length by 57.6 ± 14.7% relative to control (FIG. 3G, H). Quantitative analysis of collagen intensity from Masson's trichrome staining (FIG. 3G, I) and CD31 fluorescence intensity (FIG. 3G, J) demonstrated that HPMN treatment promoted greater collagen production by 68.7 ± 11.9% and angiogenesis by 222.0 ± 110.5%, respectively. Finally, immunohistochemical staining revealed that MCP-1 level was significantly reduced by 59.2 ± 12.5% from HPMN treatment compared to control (FIG. 3G, K). In summary, it was demonstrated that HPMN can spatially enrich inflammatory chemokines (e.g, MCP-1) and monocytes to alleviate wound inflammation and accelerate diabetic wound healing.

[0185] Due to the high physiological similarity between humans and pigs for wound healing mechanisms including dermis restoration, skin re-epithelialization, and skin contraction, pigs are an excellent large animal model to assess strategies to promote healing of wounded tissues. A porcine wound model was used to evaluate the ability of HPMN to promote wound tissue regeneration (FIG. 4A). HPMN was applied to wounds on days 3 and 7 post wounding and could extract abundant amount of MCP-1 on day 3. The extracted amount of MCP-1 on day 7 was 41.5 ± 16.5% of that extracted on day 3, which was similar to our earlier observation in mice diabetic wounds, likely attributing to depletion of inflammatory cells and termination of positive inflammation feedback loop (FIG. 9). After the pigs were euthanised on day 15, H&E-stained wound sections showed that HPMN treatment improved wound closure by 27.2 ± 5.0% compared to control (FIG. 4B, C). Immunofluorescence staining of CD31 revealed enhanced angiogenesis of 105.0 ± 32.9% for HPMN treated wounds (FIG. 4D). Further, immunohistochemical staining of Ki67, an indicator of cell proliferation, demonstrated HPMN treatment induced 35.9 + 13.7% more proliferating cells (FIG. 4E).

[0186] To understand whether HPMN could modify the immune microenvironment of wounded tissues to improve regeneration, the presence of key immune cells including macrophages, neutrophils, and T cells were examined using immunohistochemical staining (FIG. 4F-H). Wounds treated with HPMN showed significant decrease in Ibal+macrophages (34.4 ± 8.8%), neutrophils (51.2 + 6.1%), and CD3+T cells (50.6 ± 11.4%) compared to control, implying a reduced influx and presence of inflammatory immune cells. We also performed bulk tissue gene analysis to complement the histological experiment. HPMN treatment was associated with a down-regulation of pro-inflammatory genes relative to control including tumor necrosis factor (TNF), interleukin-6 (ILS), chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 4 (CCL4), C-X-C motif chemokine ligand 8 (CXCL8), and colony-stimulating factors 2 (CSF2), and these results were in strong agreement with reduced infiltration of immune cells from histological analysis (FIG. 41). On the contrary, the expression of pro-healing genes encoding growth factors such as fibroblast growth factor 2 (FGF2), platelet-derived growth factor subunit B (PDGFB), vascular endothelial growth factor A (VEGFA), transforming growth factor beta 1 (TGFB1), and insulin-like growth factor 1 (IGF1), extracellular matrix structural components such as collagen type I, III, and V (COL1A1, COL3A1, and COL5A2), fibronectin 1 (FN1), and tenascin (TNC), and remodeling enzymes such as matrix metalloproteinases (MMP2 and MMP9) and tissue inhibitors of metalloproteinase (TIMP1 and TIMP2) were up-regulated in HPMN treated wounds. Collectively, the results showed that HPMN could decrease inflammatory burden to improve angiogenesis and cell proliferation to benefit wound healing in a porcine wound model.

[0187] Example 4: Combination therapy of HPMN and immunosuppressants ameliorates psoriasis

[0188] Psoriasis is a chronic inflammatory skin disorder characterized by an abundant infiltration of immune cells. To investigate the therapeutic potential of HPMN for other inflammatory skin diseases and conditions, HPMN was combined with immunosuppressants (e.g., methotrexate (MTX)) to alleviate skin inflammation in an imiquimod-induced psoriasis model. MTX is a folic acid analog that induces apoptosis of proliferating keratinocytes, and it can be coated onto the surface of HPMN (MTX HPMN, ~ 4.7 j g of MTX per patch), with 50% of drugs being delivered into mouse skin (FIG. 10A). Of note, MTX-loaded HPMN retained the same capacity for MCP-1 binding as HPMN (FIG. 10B). Psoriasis model mice were established by treating them with imiquimod for 6 consecutive days. MTX HPMN treatment was started on day 3 when apparent skin inflammation appeared (FIG. 4A). The psoriasis area and severity index (PASI) is a clinical score that reflects the severity of erythema, scaling, and skin thickening. Repeated topical induction with imiquimod significantly increased the PASI score in model mice, while MTX HPMN therapy mitigated the increase from days 5-7 (FIG. 4B). The spleens of model psoriasis mice were enlarged with heavier weights (FIG. 4C, D), this observation was consistent with previous reports that psoriasis leads to higher total cell numbers and the release of proinflammatory cytokines. In comparison to model psoriasis mice, MTX HPMN-treated mice had significantly lighter spleen weights, indicating a dramatic reduction of inflammation.

[0189] It is known that the hyperproliferation of keratinocytes causes thickened epidermis in psoriasis. H&E-stained dorsal skin sections were examined and it was found that imiquimod-induced psoriasis model mice showed excessive thickening of the epidermis (86.2 ± 13.9 .m) compared to normal mice (19.5 ± 2.2 .m), and MTX H PM N -treated mice reduced epidermis layer by 45.1 ± 7.7% (FIG. 4E, 11). Keratinocyte hyperplasia was further assessed by immunohistochemical staining of Ki67, a nuclear proliferation indicator reflecting keratinocyte proliferation and the severity of psoriasis. There was a significant down-regulation of Ki67 with MTX HPMN treatment which is consistent with reduced skin thickness (FIG. 4F, 4G). Notably, MTX HPMN treatment significantly reduced the tissue MCP-1 level which became comparable to that in control skin tissues of healthy mice (FIG. 4H, 41).

[0190] As MCP-1 is responsible for immune cell recruitment that orchestrates and aggravates psoriasis, MCP-1 depletion from psoriasis skin may reduce immune cell influx. After MTX HPMN treatment on day 7, mice were euthanized, and immune cell populations from skin were analyzed by flow cytometry (FIG. 12, 13). MTX HPMN treatment was found to significantly reduce the monocyte population from 57.3 ± 24.0% to 15.9 ± 9.5% (FIG. 5J). As monocytes can differentiate into inflammatory dendritic cells and macrophages under inflammatory stimulation during the pathogenesis of psoriasis, MTX HPMN treatment which stems monocyte infiltration could lead to decreased numbers of macrophages (FIG. 5K) and dendritic cells (FIG. 5L) in psoriasis skin. T cell infiltration in the epidermis and the activation of T cells plays a key role in the pathogenesis of psoriasis, and MTX HPMN could also reduce their numbers. Consistent with this reasoning, a dramatic reduction in T cell influx was observed (FIG. 5M). Cytokines released by infiltrated immune cells, such as IL-17 secreted by T cells and IL-23 secreted by macrophages and dendritic cells, are known to result in the hyperproliferation of keratinocytes through the IL17 and IL23 pathway in psoriasis. There was a remarkable down-regulation of IL17a, IL23 and the pro-inflammatory ILlb gene expression after HPMN treatment when compared to model psoriasis mice (FIG. 5N). This shows that HPMN could be applied to a broad range of inflammatory skin conditions and diseases, particularly with psoriasis which has a thickened epidermis where topical drug application on the surface of skin is unlikely to stem the root cause of inflammation i.e., cells and cytokines spatially enriched, deep within skin tissues.

[0191] Thus, it is shown that porous microneedles with a coating of a StarPEG / heparin network is able to concentrate and sequester multiple chemokines, with preferential binding to inflammatory MCP-1 chemokine. With the enrichment of MCP-1, HPMN was able to recruit inflammatory monocytes through the porous structure on the microneedle surface. Depletion of chemokines and monocytes from inflammatory sites attenuated tissue MCP-1 level and reduced monocyte infiltration in diabetic wounds, which resulted in accelerated diabetic wound healing due to a shortened inflammatory duration. Reduced immune cell infiltration was also observed in porcine wounds after HPMN treatment. Besides application in wound healing, HPMN can be extended to alleviate inflammation in psoriasis by combining it with existing immunosuppressants like MTX to improve PASI score and reduce epidermal thickness.

[0192] HPMN's ability to penetrate tissues to sequester chemokines and monocytes is an improvement over current methods of treating wounds. GAG-based hydrogels are limited to interacting with chemokines only at the wound surface or in wound exudates. In contrast, HPMN can access thicker wounds to sequester chemokines deep within the wound tissues, which is also useful for treating other inflammatory skin conditions such as psoriasis, where superficial application of therapeutics are not likely to be effective. Another advantage is that HPMN provides a large surface area for chemokine binding. HPMN has a 1.9-fold higher surface area theoretically that could enable it to interact with wounds better than the patch without needles (HPP) (calculated from nonporous patches). FIG. 3B showed that HPMN extracted 1.6-fold higher MCP-1 than HPP on day 3. Another advantage of HPMN is that it is capable of capturing inflammatory monocytes as shown in vitro by monocyte infiltration (FIG. 2G) and in vivo by decreased numbers of monocytes at the wound site (FIG. 3D). This is crucial as monocytes are the main source of chemokines and the root cause of chronic inflammation. The primary materials used to make HPMN are PLGA and heparin, which are biocompatible and already in clinical use. Further improvements in microneedle design, e.g., by using degradable or enzymatically digestible materials, may facilitate cell recovery from the microneedles for analysis, and allow longitudinal tracking of changes in immune subtypes during wound progression. Advances in additive manufacturing can also allow HPMN patches of different shapes and sizes to be custom-fabricated for individual patients. Combination therapy of HPMN with antibiotics can prevent biofilm formation and reduce complications from infections. Finally, as chronic human wounds usually last for more than 4 weeks and is accompanied by prolonged inflammation, the dressing frequency of HPMN can be appropriately optimized for better inflammation control.

[0193] Table 1. Porcine primer sequences for RT-PCR assay.

[0194] Table 2. Mouse primer sequences for RT-PCR assay.

[0195] Hydrogel coated porous microneedle

[0196] Gelatin type A was dissolved in DMSO, after obtaining a homogeneous solution, di-tert-butyl dicarbonate (Boc) was added and the reaction mixture was stirred for 80 min. Mixture was precipitated in a 10-fold excess of cold acetone. The residue was dissolved in water and dialyzed against water. The dialyzed Gelatin- Boc solution was placed in a 250 mL flask, EDC and NHS were added and stirred for 30 min, then cystamine dihydrochloride were added and reacted for 24 h. After that, the mixture was dialyzed against water for 2 days, filtered, and freeze-dried to obtain cystamine-conjugated gelatin. The cystamine-conjugated gelatin was dissolved and stirred in PBS solution, and subsequently, methacrylic anhydride (MA) was added dropwise and stirred in dark. After a 4-hour reaction, the mixture was dialyzed for 2 days and freeze-dried to obtain GSSMA.

[0197] Hydrogel coating on microneedles: GSSMA was dissolved in 0.2% photoinitiator (LAP) water solution to a concentration of 30%, dextran was dissolved in water to 20%-40%. GSSMA and dextran was mixed and add to the PLGA microneedle substrate, after UV crosslinking for 5 min, the coated microneedles were washed with water for 2 hours to remove dextran, micropores were generated after the removal of dextran.

[0198] A hydrogel formed from Gelatin-Boc-Cystamine-MA (GSSMA) is coated on the microneedle. The hydrogel is UV crosslinkable and degradable, and the pore size on coating layer is controllable by adjusting the mass ratio of GSSMA and dextran. By adjusting the pore size, the cell extraction properties in vitro may be optimized. After cell extraction, the coating hydrogel can be degradable in the presence of reducing agent such as TCEP (tris(2-carboxyethyl)phosphine) to recover cells for further analysis.

[0199] Figure 12 shows H-NMR characterization of synthesized Gelatin-Boc-Cystamine- MA (GSSMA). Figure 13 shows degradation properties of GSSMA in the presence of different concentration of TCEP. Figure 14 shows pore size of GSSMA hydrogel can be turned by using various dextran concentration.

[0200] Figure 15 shows GSSMA coated microneedles extracted and recovered murine macrophages (RAW 264.7) using an in vitro GelMA hydrogel model.

Claims

Claims1. A microneedle comprising: a glycosaminoglycan (GAG) coating and / or a hydrogel layer on an external surface of the microneedle; wherein the microneedle and / or the hydrogel layer is porous.

2. The microneedle according to claim 1, wherein the GAG coating further comprises a polymer, wherein GAG is covalently bonded to the polymer.

3. The microneedle according to claim 1 or 2, wherein the GAG coating is a bilayer coating, comprising a first polymer layer adjacent to the microneedle, and a second layer comprising GAG covalently attached to the first polymer layer.

4. The microneedle according to any one of claims 1 to 3, wherein the polymer is star polyethylene glycol (PEG).

5. The microneedle according to any one of claims 1 to 4, wherein the GAG is heparin.

6. The microneedle according to any one of claims 1 to 5, wherein the GAG is characterised by a concentration of about 1 mg / mL to about 5 mg / mL relative to the GAG coating.

7. The microneedle according to any one of claims 1 to 6, wherein the microneedle comprises poly(lactic-co-glycolic acid) (PLGA).

8. The microneedle according to any one of claims 1 to 7, wherein the porous microneedle is characterised by a pore size of about 5 pm to about 50 pm.

9. The microneedle according to any one of claims 1 to 8, wherein the microneedle is characterised by a length of about 500 pm to about 1500 pm, and / or a base width of about 400 pm to about 600 pm.

10. The microneedle according to any one of claims 1 to 9, wherein the microneedle further comprises an active substance selected from an immunosuppressant, an antimicrobial compound, an antimetabolite, or a combination thereof.

11. The microneedle according to any one of claims 1 to 10, wherein the microneedle is capable of recruiting monocyte chemoattractant protein-1 (MCP-1) and / or monocyte or macrophage within its pores thereof.

12. A microneedle device, comprising a plurality of microneedles extending from a substrate, wherein one or more of the microneedles is a microneedle according to any one of claims 1 to 11.

13. The microneedle device according to claim 12, wherein the plurality of microneedles is present as an array on the substrate, wherein the distance between two microneedles on the microneedle array is about 500 pm to about 1000 pm.

14. The microneedle device according to claim 12 or 13, wherein the substrate is attached to a dressing.

15. The microneedle device according to claim 14, wherein the dressing comprises an adhesive for attaching to a tissue.

16. A method of treating a wound and / or a skin disease or condition in a subject in need thereof, the method comprising applying a microneedle device according to any one of claims 12 to 15 to the wound and / or skin havingthe skin disease or condition such that one or more of the microneedles in the device penetrates the wound or skin.

17. The method according to claim 16, wherein the porous microneedle recruits a cytokine and / or a motile cell from the wound.

18. The method according to claim 16 or 17, wherein the wound is a chronic wound.

19. The method according to any one of claims 16 to 18, wherein the wound is a wound on a skin tissue.

20. The method according to any one of claims 16 to 19, wherein the skin disease or condition is an inflammatory skin disease or condition.

21. The method according to any one of claims 16 to 20, wherein the skin disease or condition is psoriasis.

22. The method according to any one of claims 16 to 21, further comprising contacting the microneedle device to the wound and / or skin disease or condition for at least about 12 h.

23. A method of isolating a chemokine and / or motile cell from a tissue from a subject, the method comprising (a) applying a microneedle array according to any one of claims 12 to 15 to the tissue such that one or more of the porous microneedles in the device penetrates the tissue; and (b) removing the microneedle array from the tissue after a time sufficient for the porous microneedle to recruit the chemokine and / or motile cell from the tissue, thereby isolating the recruited chemokine and / or motile cell from the tissue.

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